Production and Purification of pre-miRNA-149 with potential application in diagnosis of prostate cancer

• Autor(a) principal: Martins, Henrique Samuel Ramos
• Data de Publicação: 2018
• Tipo de documento: Dissertação
• Idioma: eng
• Título da fonte: Repositório Científico de Acesso Aberto de Portugal (Repositórios Cientìficos)
• Texto Completo: http://hdl.handle.net/10400.6/6990
• topic: Cromatografia de Afinidade.; G-Quadruplex; Pre-Mirna-149; Purificação; Domínio/Área Científica::Engenharia e Tecnologia::Biotecnologia

A few years ago, was discovered that the lin-4 gene did not encode a protein, but a small RNA, responsible for regulating the expression of the LIN-14 protein in Caenorhabditis elegans. This promotes the study of a new class of biomolecules, the non-coding RNAs (ncRNAs), which include the microRNAs (miRNAs). Among them, the miRNA-149 is particularly relevant in the several cellular processes, including oncogenic regulators. Another particularity of this molecule is that the precursor (pre-miRNA-149) have a G-rich sequence, which forms a special nucleic acid secondary structure called G-quadruplex (G4) with a parallel topology that allows its recognition by nucleolin (an important nucleolar protein). This structure has been associated to the regulation of many cellular processes, such as, telomere maintenance, replication, transcription and translation. The present work describes the production and purification of pre-miRNA-149 in E. coli DH5a. For that purpose, a pre-miRNA-149 DNA sequence was cloned into a pBHSR1-RM plasmid and inserted within the previous competent cells by thermic shock. After fermentation, total RNA was and purified by affinity chromatography taking advantage of the biological recognition of pre-miRNA-149. Three affinity supports are used such as naphthalene amine Sepharose, Ltyrosine Sepharose and L-lysine Sepharose. Each of them has already been successfully used to purify similar biomolecules. The eluted fractions are evaluated by denaturing PAGE. The complete isolation of pre-miRNA-149 was not achieved in the three supports tested; however, some promising results were obtained with the L-lysine support, where a small portion of pre-miRNA-149 was isolated into one single fraction (low recovery rate), with a decreasing stepwise gradient from 2.05 M to 0.1 M of (NH4)2SO4 in Tris 10 mM (pH 6.0). Future affinity chromatography experiments are required to optimize the conditions to separate the pre-miRNA-149 from the total RNA mixture, to successfully use miRNA-based therapies.